Recently vascular disorder including atherosclerosis is rising with the increase of adult diseases. One of the representative diseases of vascular disorder is atherosclerosis, which is the hardening of artery, in relation with lipid metabolism, attributed to various environmental causes such as dietary habits, smoking, lack of exercise, etc. and mostly developed in cerebral artery or coronary artery and further progressed to circulatory system disease such as heart disease and cerebrovascular disease. For instance, cerebral atherosclerosis shows such symptoms as headache, dizziness or mental disorder and might be progressed to encephalomalacia. Coronary atherosclerosis causes a pain in the heart and arrhythmia, which might be responsible for angina pectoris and myocardial infarction. Atherosclerosis also causes hypertension, heart disease, and apoplexy, etc, and thus atherosclerosis related diseases particularly become one of leading causes of death among men at the age of 50-60s in modern society.
The primary outbreak of atherosclerosis is outlined by ‘response-to-injury hypothesis’ containing the explanation on chronic inflammation on the damage on blood vessel wall [New Engl. J. Med. 1999, 340, 115-126]. That is, the lost of homeostasis and mal-functioning in vascular endothelial cells, which are attributed to genetic variation, peroxides, hypertension, diabetes, the increase of plasma homocysteine content or/and microorganism infection, result in arteriosclerosis.
More precisely, by the above reasons, low-density lipoprotein (LDL) is converted into highly-modified LDL (HM-LDL) through oxidation, glycosylation, integration, glycoprotein binding, etc, resulting in the stimulation and damage on vascular endothelial cells and smooth muscle. Subsequently, the expression of vascular cell adhesion molecule-1 (VCAM-1) and release of inflammatory mediator of inflammatory cells in endothelial cells are accelerated, by which LDL flows in and is accumulated in endothelial cells. The accumulated LDL and oxidized HM-LDL promote the inflow and activation of immune cells such as macrophages and T-lymphocytes, resulting in the continuous inflammatory reaction on lesions. Then, the in-coming macrophages or lymphocytes release a hydrolase, an inflammatory mediator and a growth factor, which would destroy the lesion. Then, monocytes flow in and smooth muscle cells are migrated and differentiated and fibrous lesions are generated in the necrotized lesion area. Through the repeated procedure above, the lesions are developed into the fibrous plaque with complicated structure in which necrotizing tissue containing HM-LDL is covered with fibers. Macrophage secretion of matrix metalloproteinases and neovascularization contribute to weakening of the fibrous plaque. Plaque rupture exposes blood components to tissue factor, initiating coagulation, the recruitment of platelets, and the formation of a thrombus. The thrombus and artery hardening lead to the cardiovascular disease including vascular insufficiency.
LDL oxidation is believed to be the most responsible primary cause of arteriosclerosis including atherosclerosis [Circulation, 1995, 91, 2488-2496; Arterioscler. Thromb. Vasc. Biol., 1997, 17, 3338-3346]. Oxidative stress generated in vivo or ex vivo converts LDL into oxidized-LDL. Monocytes attach to endothelial cells that have been induced to express cell adhesion molecules by oxidized-LDL and inflammatory cytokines. Adherent monocytes migrate into the subendothelial space and differentiate into macrophages. Uptake of oxidized LDL via scavenger receptors leads to foam cells, resulting in the generation of fatty streak which is the primary lesion of atherosclerosis. The primary lesion of atherosclerosis is characterized by the expressions of adhesion molecules VCAM-1, ICAM-1 (intracellular adhesion molecule-1) and MCP-1 (monocyte chemoattractant protein-1), which are generated in arterial endothelial cells. The expressions of such adhesion molecules are induced by NF-κB (nuclear factor-κB), a transcription factor. NF-κB also causes plaque formation and rupture on blood vessel. NF-κB is activated by various factors including reactive oxygen species (ROS) and cytokine, and is a hetero dimmer composed of p50 and p65 which is included in cells as a transcription factor to regulate many types of target genes. The activated NF-κB is linked to a specific promoter gene to regulate the expression of various inflammatory factors such as IL-1, VCAM-1, ICAM-1 and other factors involved in the progress of atherosclerosis.
It has been reported that antioxidants and radical scavengers inhibit NF-κB activity. Thus, it is expected that an antioxidant inhibits LDL oxidation and adhesion molecule expression, decrease NF-κB activity and thereby arrests atherosclerosis in vivo. And to confirm the expectation, studies have been going (Korean Patent Publication No. 2003-0014155). Besides, studies to find out a LDL peroxide forming factor and eliminate thereof from patients with hyperlipidemia and atherosclerosis [Curr. Atheroscler. Res., 2000, 2, 363-372].
Cytokines are involved in various physiological and pathological processes, and particularly play an important role in immune response, inflammation, tissue reorganization and blastogenesis (Akoum et al., Hum. Reprod. 11:2269-2275, 1996; Inadera et al., Endocrinology 141:50-59, 2000; Xu et al., Life Sci. 64:2451-2462, 1999). Among many cytokines, IL-1α, IL-1β, IL-6, TNFα, and IFNγ are deeply involved in infection or tissue wound (Akoum et al., Hum. Reprod. 11:2269-2275, 1996; Danforth and Sgagias, J. Endocrinol. 138:517-528, 1993; Deshpande et al., Am. J. Reprod. Immunol. 38:46-5, 1997). These cytokines act as intracellular pyrogens and have been also called as inflammatory cytokines (Angele et al., J. Physiol. 277:C35-42, 1999; Bradley and Timothy, Environ. Toxicol. and Chem. 17:3-14, 1998; D'Agostino et al., Ann. N.Y. Acad. Sci. 876:426-429, 1999; Galien and Garcia, Nucleic Acids Res. 25:2424-2429, 1997).
Nitric Oxide (NO) is one of products by inflammatory cytokine in immune system. NO is normally secreted in mammaria, which plays an important role in vascular tone regulation, platelet function, neurotransmission and host defense mechanism (Zancan et al., Endocrinology 140:2004-2009, 1999). In general, only a small amount of NO is generated in some cells (endothelial cells, neurons) but Ca+-independent NO synthase is induced by lipopolysaccaride (LPS) in many kinds of cells such as macrophages, neutrophils, Kupffer's cells and stem cells (Marietta et al., Biochemistry, 27:8706-8711, 1988; McCall et al., Br. J. Pharmacol., 102: 234-238, 1991; Curran et al., J. Exp. Med., 170: 1769-1774, 1989), in particular significant amount of inducible NO synthase (iNOS) is detected in macrophages (Hiki et al., Jpn. J. Pharmacol., 56: 217-220, 1992; Lowenstein et al., Proc. Natl. Acad. Sci. USA 90:9730-9734, 1993).
Macrophages are very important factor which plays an important role in host defense mechanism by engulfing various tumor cells or microorganisms or inhibiting the proliferation thereof when they are activated by foreign substances. It is reported that NO is involved in cell lysis of macrophages (Hibbs et al., Science, 235, 473-476, 1987). When macrophages are activated by such cytokine as IL-1α or LPS, they lead the expression of iNOS and then the iNOS catalytes NO synthesis from L-arginine and oxygen molecules (Palmer et al., Nature 333:664-666, 1988; Karupiah et al., Science 261:1445-1448, 1993; Kleemann et al., FEBS Lett. 328:9-12, 1993; Wong et al., Adv. Pharmacol. 34: 155-170, 1995). Macrophages also promote T-cell immune response by antigen-presenting and generating cytokine like IL-1α. In addition to normal immune response, iNOS and IL-1α induce inflammatory reaction when they are over-expressed, indicating that regulation of gene expression is a crucial factor to control immune or inflammatory reaction (Wong et al., Adv. Pharmacol: 34: 155-170, 1995; Evans, Agents Actions Suppl. 47: 107-116, 1995; Vane et al., 1994; Moilanen et al., Am. J. Pathol. 150: 881-887, 1995).
The regulation of gene expression by the inflammatory cytokine is mostly conducted during the transcriptional stage of the gene. Particularly, the expression is regulated by the DNA-binding protein such as a transcription factor which recognizes and interacts with the promoter and enhancer element of a gene (Nill et al., J. Immunol. 154:68-79, 1995). It has been known that four transcription factors ‘CREB, AP-1, NF-IL6 and NF-kB’ are involved in transcriptional regulation of the inflammatory cytokine (Cippitelli et al., J. Biol. Chem. 270: 12548-12556, 1995; Dendorfer, Organs 20:437-44, 1996; Geist et al., Am. J. Reapir Cell. Mol. Biol. 16: 31-37, 1997; Shenkar and Abraham, Am. J. Reapir Cell. Mol. Biol. 14:198-206, 1996; Xie et al., J. Biol. Chem. 269:4705-4708, 1994). Specifically, the expressions of iNOS and IL-1α are known to be regulated by the extent of NF-κB activity (Xie et al., J. Biol. Chem. 269:4705-4708, 1994).
Another responsible factor for coronary heart disease is high blood cholesterol. So, it is important to lower the level of blood cholesterol by dietary treatment reducing cholesterol and lipid intake or by inhibiting cholesterol absorption by suppressing, lipid metabolism-related enzymes. To this end, studies on acyl-CoA:cholesterol acyltransferase (ACAT) which is an enzyme responsible for cholesterol estherification have been undergoing.
ACAT is functioning largely in the intestines, the liver and blood vessel wall cells.
First, ACAT esterifies cholesterol in the liver and promotes the absorption thereof. Second, cholesterol, either taken in or synthesized in vivo, is accumulated in very low-density lipoprotein (VLDL), a carrier, in the liver, and then migrated to each organ through blood vessels. At this time, the accumulation of cholesterol in the carrier is made possible by the estherification of cholesterol that is cholesterol is converted into cholesteryl ester. Third, ACAT converts, cholesterol into cholesteryl ester in arterial vessel wall forming cells to promote the accumulation of intracellular cholesterol, which is direct cause of atherosclerosis. By the activation of ACAT, foam cells are subject to contain high level of cholesteryl ester converted from cholesterol. Thus, foam cell formation in macrophages and smooth muscle cells is very important in clinical and experimental aspects. The foam cell proliferation in the vessel wall is closely related to the activation of ACAT, making an ACAT inhibitor as a promising candidate for an effective anti-atherosclerotic agent.
Therefore, it is expected for an ACAT inhibitor firstly to reduce the cholesterol level by inhibiting the absorption of cholesterol in the intestines, secondly to lower the blood cholesterol level by inhibiting the release of cholesterol, migrated from the liver, in blood vessels and thirdly to prevent directly atherosclerosis by preventing the accumulation of cholesterol in the vessel wall.
ACAT inhibitors known so far are inhibitors of mouse liver microsomal ACAT or mouse macrophages (J774) ACAT. Human ACAT was found to be present as two isoforms, ACAT-1 (50 kDa) and ACAT-2 (46 kDa). ACAT-1 is found primarily in the Kupffer cells of the liver, adrenal cortical dells, macrophages, and kidney, while ACAT-2 is mainly located in hepatocytes and intestinal mucosal cells [Rudel, L. L. et al., Curr. Opin. Lipidol. 12, 121-127, 2001]. The human ACAT inhibitors inhibits, the absorption of cholesterol taken from food and accumulation of cholesteryl ester in the vessel wall, making it a successful candidate for a preventive and therapeutic agent for hypercholesterolemia, cholesterol gallstone, or atherosclerosis [Buhman, K. K. et al., Nature Med. 6, 1341-1347, 2000].
Recent hyperlipidemia therapeutic agents such as probucol, N,N′-Diphenylenediamine, butylated hydroxy anisol (BHA) and butylated hydroxy toluene (BHT), phenol synthetic antioxidants, have excellent antioxidant activity, so that they reduce the level of LDL cholesterol, arrest oxidation and reduce lesion development but have side effects too.
To prevent such diseases without side effects, an attempt has been made to reduce the level of LDL by inhibiting the absorption and synthesis of cholesterol [Principles in Biochemistry, lipid biosynthesis, 770-817, 3rd Edition, 2000 Worth Publishers, New York; Steinberg, N. Engl. J. Med., 1989, 320, 915-924]. Accordingly, the possibility of combined treatment of LDL-antioxidant and lipid lowering agent has been a major concern to treat patients with hyperlipidemia or atherosclerosis.
The centipede has been utilized as a medicinal animal. And, a method for isolating and purifying a thrombolytic enzyme from large centipede (Scolopendra subspinipes multilans) is described in Korean Patent No. 124438. Compounds isolated from the centipede are 3,8-dihydroxyquinoline [Moon, et al., J. Nat. Prod. 59: 777-779, 1996], 2-hydroxy-7-[(4-hydroxy-3-methoxyphenyl)methyl]-3-methoxy-8-quinolyl sulfate [Noda, et al., Chem. Pharm. Bull. 49(7): 930-931, 2001], and 8-hydroxy-1H-2-benzopyran-1-one [Kim, et al., J. Korean Chem. Soc. 42: 236-239, 1998]. However, there has been no report, yet, on the results of study on antioxidant effect or cholesterol metabolism-associated activity of the centipede and utilization of the centipede as a cardiovascular disease treating agent.
In the course of searching for a preventive and therapeutic agent for cardiovascular diseases including hyperlipidemia, atherosclerosis, coronary heart disease, and myocardial infarction from natural substances, the present inventors found out that the centipede extracts and quinoline and phenol compounds isolated therefrom exhibit excellent LDL-antioxidant, ACAT inhibitory, and anti-inflammatory activity. And further, the present inventors completed this invention by confirming that the above centipede extracts and compounds isolated from the same can be effectively used for the prevention and treatment of cardiovascular diseases such as hyperlipidemia, atherosclerosis, coronary heart disease, and myocardial infarction.